Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney.

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.     

Guanylate cyclase activity of human lymphocytes from peripheral blood, thymus, and tonsils. A comparative study

The subcellular repartition and the distinctive properties of guanylate cyclase (EC 4.6.1.2) vary according to the lymphocyte population studied and according to the presence of detergent. Guanylate cyclase of non-adherent peripheral lymphocytes and of thymus lymphocytes is recovered by more than 90% in the soluble fraction of the homogenate. Kinetics according to the substrate (5'-GTP-Mn2+) is Michaelian, the Ca2+ ion acts as an activator, especially in the case of blood lymphocytes, and the detergent has no effect on the enzyme activity. On the other hand, the guanylate cyclase of tonsil lymphocytes reside in the particulate fraction. It has non-Michaelian kinetics for the substrate, a strong stimulating effect of detergent, and an inhibitory effect of Ca2+. A comparison of the enzymatic activities of unseparated and of non-adherent tonsil lymphocytes obtained from the same donor points to a correlation between their T and B properties: predominant soluble activity in the T population and particulate guanyl cyclase activity in the B subset.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

Isolation of the outer acrosomal membrane from bull sperm.

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.     

Distribution subcellulaire des protéine-kinases stimulables par l'AMP cyclique et des protéines réceptrices de l'AMP cyclique dans la thyroïde de porc

The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent.Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent.Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes.The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions.     

Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification

Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

Solubilization of frog brain and retina cholinesterase and studies of different molecular forms.

1. The cholinesterase (ChE) of frog brain and retina could be easily solubilized. About 10% of the brain and 20% of the retina ChE were found to be soluble in 0.05 M phosphate buffer. After treatment with 0.5% (v/v) Triton X-100, about 30% of the total ChE activity of the brain and only 10% for retina was left particle bound. NaCl by itself did not solubilize ChE. Use of higher NaCl concentrations in combination with Triton X-100 as well as higher detergent concentrations alone seemed to cause an inhibiting effect of the solubilized ChE from retina. 2. The solubilized ChE from brain as well as retina were electrofocused as one main activity peak, corresponding to isoelectric points of pH 6.1 and 6.0, respectively. A second molecular form at pH 5.9 was distinguishable for the brain, but not for retina ChE. 3. Sucrose gradient centrifugation indicated that the ChE solubilized from the brain and retina consists of two molecular forms exhibiting S values of 5.1 +/- 0.24, 10.9 +/- 0.33 and 6.1 +/- 0.30, 10.9 +/- 0.43, respectively. After solubilization by higher Triton X-100 concentrations the soluble extracts from brain and retina seemed to contain the activity of these forms in different proportions. 4. Polyacrylamide gel electrophoresis separated three molecular forms of the brain ChE. One of these forms was found to have a molecular weight of 394,000 +/- 20,000. The others were found to have an identical molecular weight of 550,000 +/- 10,000. Two molecular forms exhibiting molecular weights of 292,000 +/- 10,000 and 470,000 +/- 10,000, could be separated for retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution of hepatic bile acid-conjugating enzymes.

1. The subcellular location of enzymes conjugating bile acids with glycine or taurine was investigated by centrifugation of rat liver homogenates. 2. [14C]Cholic acid-conjugating activity was predominantly associated with the soluble-microsomal region of the gradient after centrifugation in a Ti-15 zonal rotor but the bulk of the conjugating activity sedimented with mitochondrial-lysosomal fractions in differential pelleting experiments. 3. Cholate: CoA ligase (EC 6.2.1.7) and cholyltransferase (EC 2.3.1) were not enriched in purified Golgi or plasma-membrane fractions. Cholate: CoA ligase was distributed evenly between rough- and smooth-surfaced microsomal subfractions but cholyltransferase showed a dual soluble-rough microsomal activity distribution. 4. Sedimentation of cholyltransferase in mitochondria-enriched fractions prepared by differential centrifugation appears to be an artefact of sedimentation of rough microsomal membranes in mitochondrial fractions. 5. The subcellular distribution of bile acid-conjugating enzymes is discussed with reference to hepatic processing of bile acids.     

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.     

Brain enzyme activity in irradiated sympathectomized rats

The results are reviewed from studies of activity of hexokinase (2.7.1.1.EC), dehydrogenase glucose-6-phosphate (1.1.1.49 EC), and cholinesterase (3.1.1.7 EC) in subcellular fractions of rat brain at the background of chemical sympathectomy induced by long-term administration of guanethidine and subsequent irradiation with a dose of 7 Gy. In conditions of sympathectomy, the enzyme activity is inhibited; in irradiated sympathectomized rats, activity of hexokinase and cholinesterase increases to reach the level of that of intact animals while dehydrogenase remains inhibited.     

Soluble precursor of membrane-associated protein kinase in uterine smooth muscle cells

Incubation of smooth muscle strips from rat uterus with isoproterenol resulted in redistribution of protein kinase activity between the cytosol and a 20,000 to 50,000g membrane fraction. Similarities in the elution properties of the cytosolic and membrane-associated forms of the enzyme on DEAE-cellulose ion exchange chromatography further suggested the two forms were the same. The nature of membrane binding of the soluble enzyme was investigated using smooth muscle microsomal and cytosol fractions. Membranes readily bound the soluble enzyme when the two subcellular compartments were reconstituted and incubated at 30 °C for 10 min. The extent of binding was proportional to the ratio of membranes to cytosol and was characterized by the inhibition of soluble enzyme activity toward exogenous substrates in a Triton X-100 reversible manner. In marked contrast to the binding of soluble protein kinase to heart particulate fractions, binding of the cytosol enzyme to smooth muscle cell membranes was unaffected by ionic strength or cAMP. The latter property indicated holoenzyme was bound in a manner similar to the free catalytic subunit of cAMP-dependent protein kinase and suggested the enzyme was bound by association between the membrane and the catalytic subunit. Binding of cytosol protein kinase to the membranes rendered the enzyme insensitive to trypsin digestion and the capacity of the smooth muscle cell membranes to bind the soluble enzyme exceeded that of other rat tissue fractions. Resistance to salt extraction and proteolysis, as well as its detergent dependence, suggested the soluble enzyme became an integral or intrinsic membrane protein following association with the membrane. The ability of membranes to incorporate [γ-³²P]ATP into phosphoprotein was lost on detergent extraction of protein kinase and restored in an apparently specific manner when extracted and washed membranes were reconstituted with soluble enzyme. The intrinsic nature of membrane protein kinase and the apparent specificity with which the soluble enzyme was hound by membranes further indicated that, in myometrium. hormone-induced translocation of protein kinase is an important mechanism by which enzyme activity is increased in the vicinity of its in situ substrates.     

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.     

Acetylcholinesterase of Schistosoma mansoni. Molecular forms of the solubilized enzyme

Several molecular forms of acetylcholinesterase were obtained from Schistosoma mansoni homogenates by extraction in either low-salt buffer, high-salt buffer or detergent buffer. The low-salt soluble form amounts to 25% of the total activity. By contrast, the extract obtained in the presence of Triton X-100 possessed almost almost 3-fold higher enzymatic activity, most of it (86%) being retained in the soluble extract (100 000 X g). High-salt concentration (1 M NaCl) also has a solubilizing effect, but to a lesser extent (50%). Acetylcholinesterase can also be solubilized by treatment with a solution of 1% methylmannoside (40%). In the presence of non-ionic detergents, the enzyme behaves as monodisperse 8 S form. In the absence of detergent the low-salt soluble extract is polydisperse: it contains a 10 S and a 32 S component, the latter could represent high polymers. The molecular form released from tissue homogenate by treatment with alpha-methylmannoside is polydisperse: it contains a major 10 S and a minor 32 S component. Differences in sedimentation coefficient were observed among the enzymes extracted with detergent from the various life cycle stages of the parasite. The enzyme from the cercarial stage sediments as a single 8 S peak. The adult worm exhibits an additional acetylcholinesterase peak of 18 S representing approx. 30% of the total enzymatic activity. The molecular weight of the major 8 S species, as determined by gel filtration, is 450 000.     

Acetylcholinesterase from the Skeletal Muscle of the Lamprey Petromyzon marinus Exists in Globular and Asymmetric Forms

To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subcellular distribution of acetylcholine, choline acetyltransferase and cholinesterases in lobster walking leg nerves

—Nerve bundles from the walking legs of the lobster have been homogenized in either isosmotic or hyperosmotic sucrose solutions and subjected to differential and discontinuous density gradient centrifugation. The resulting subcellular fractions were analysed for their concentrations of acetylcholine (ACh), choline acetyltransferase (ChAc) and cholinesterase (ChE). Enzymic characteristics were used for a biochemical identification of the subfractions. Regardless of the osmotic conditions, ACh was always found in the free form; there was no evidence of any‘bound’ester. After ultracentrifugation of homogenates in the hyperosmotic medium a pellet floating atop was formed; it consisted of membrane fragments and contained more than 30 per cent of the choline-esterhydrolysing enzymes, with a 3 to 4-fold increased specific activity. ChAc was found to be increasingly soluble if the ionic concentration was raised to that of the haemolymph body fluid of the lobster. Thus, all the components of the ACh system were present in substantial amounts but the question of their physiological function remains open.     

Isolation of the outer acrosomal membrane from bull sperm

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction.Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both α-fucosidase and β-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.     

The subcellular distribution and partial characterization of cholinesterase activities of canine platelets

The multiple cholinesterase activities in canine platelets have been investigated. Platelets were homogenized by rapid decompression under nitrogen, glass tube/Teflon pestle, and glycerol lysis techniques. Rapid decompression under nitrogen technique was found to be the most efficient and gentle method for cell disruption. Homogenates were subfractionated using sodium diatrizoate density gradients. Marker enzyme assays and pulse labeling experiments with 5-hydroxy[¹⁴C]tryptamine and [¹²⁵I]thrombin on prepared subcellular fractions confirmed that the soluble, plasma membrane and the granule-1 fractions were all in reasonably pure form. Furthermore, labeling of the plasma membrane with [¹²⁵I]thrombin is cited as the first successful attempt at attaining a significantly bound marker for this structure. Cholinesterase activity distributions measured in these fractions indicated that about 30% of the activity was present in the plasma membrane, 50% in granule-1 and 5% in soluble fractions. Kinetic data of cholinesterase activities obtained from intact platelets, plasma membrane preparations and platelet release supernatants indicated that they are strikingly similar.     

Distribution in spleen subcellular organelles of sialidase active towards natural sialogylcolipid and sialoglycoprotein substrates.

A procedure was devised for the preparation of enriched populations of subcellular organelles from homogenized bovine spleen. The fractions obtained were characterized for arylsulfatase, succinate dehydrogenase, UDPgalactosyltransferase and 5'-nucleotidase activities. The distribution of sialidase (acylneuraminyl hydrolase, EC 3.2.1.18) activity directed towards either endogenous substrate or exogenous ganglioside substrate suggests that it is enriched in the plasma membrane/microsomal fractions. Sialidase activity towards exogenous sialoglycoproteins, isolated from erythrocyte membrane, was enriched in the least dense of the plasma membrane/microsomal-containing fractions. The endogenous sialidase substrates were primarily the sialoglycolipids, hematoside and disialogangliosides. At the pH optimum, 3.8, and 37 degrees C, release of endogenous sialic acid was linear with time for 3 h. At the end of this time, 85% or more of the available endogenous substrate was hydrolyzed.     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.